JP2020118691A - Microscopy system, and method for operating microscopy system - Google Patents

Abstract

To provide a microscopy system and method for operating the microscopy system.SOLUTION: A microscopy system according to the present invention includes: a microscopy optical unit (14) that image-forms an object plane onto an image plane (19) through an observation beam path (22); an observation filter (23) that is in the observation beam path (22); at least two light sources that have one in two light sources provided for exciting a fluorescent dye in the object and have other provided for visualizing a non-fluorescent region of an object; and a controller that is configured to control the light sources individually. As a result of a proper configuration of the observation filter (23), all light sources can be operated with a minimum operating current, and thereby stability of light to be generated by the light source can be secured. Further, thanks to individual configurability of the light source, color rendering of the non-fluorescent region can be set. What is more, the light source can be set so that fluorescent and non-fluorescent regions appear almost equally bright.SELECTED DRAWING: Figure 1

Description

The present invention relates to microscopy systems and methods of operating microscopy systems. In particular, the microscopy system and method is useful for simultaneously observing the fluorescent and non-fluorescent regions of an object mixed with a fluorescent dye such as protoporphyrin IX (PpIX) or fluorescein.

Microscopy systems for simultaneously or sequentially observing fluorescent and non-fluorescent regions of an object are used, for example, in the field of tumor surgery. When the fluorescent dye is added to the neoplastic tissue, it binds to the neoplastic tissue. However, fluorescent dyes do not bind to non-neoplastic tissues. After exciting the fluorescent dye, an image of the tissue is recorded by fluorescence, and the fluorescent region in the tissue is identified, whereby the position of the neoplastic tissue can be specified.

In order to be able to remove the neoplastic tissue from the healthy tissue surrounding the neoplastic tissue, the neoplastic tissue must be identified and localized with respect to its surrounding tissue. Therefore, it is also necessary to capture the surrounding tissue, that is, the non-fluorescent region of the object.

To achieve this, conventional microscopy systems for simultaneously observing the fluorescent and non-fluorescent regions of an object use a white light source, such as a xenon lamp, that has a spectrally nearly uniform intensity within the visible wavelength range. Has been done. The setting of the wavelength dependent intensity of the illumination light supplied to the object is usually set by one or a plurality of illumination filters. The light emitted from the object contains both fluorescence and light reflected in the non-fluorescent region, and is usually filtered by an observation filter and supplied to an observer.

The ratio between the intensity of the fluorescence and the intensity of the light used to excite the fluorescent dye is so small that the fluorescence and the light reflected in the non-fluorescent regions of the object have a significantly different brightness. As a result, which makes it more difficult to locate the fluorescent regions and thus the neoplastic tissue. Furthermore, the intensity of fluorescence emitted from low-grade tumors is as small as 1/20 of the maximum as compared with the intensity of fluorescence emitted from high-grade tumors, which is another obstacle to localization.

Moreover, color rendering of non-fluorescent areas is often characterized by a dominant color rather than true color, which results in non-fluorescent areas being presented to the surgeon in unnatural colors. , This becomes an orientation-specific obstacle in the non-fluorescent region. This rendering, which is not true color, is due, in part, to the fact that it is technically unfeasible to improve the wavelength-dependent transmission profile of the filter, for example as an interference filter.

In view of this background, an object of the present invention is to provide a microscope inspection system, a method for operating the microscope inspection system, and a microscope inspection method that solve the above-mentioned problems.

First Aspect According to a first aspect of the present invention, a microscopy system for simultaneously observing fluorescent and non-fluorescent regions of an object is configured to image the object plane to an image plane through an observation beam path. A microscopy optics unit, an observation filter positionable in the observation beam path, at least two (narrowband) light sources, and a controller for individually controlling one or more of the at least two light sources. A controller, wherein the ratio of the intensities of the light produced by the at least two light sources is variable.

The image detector can be arranged in the image plane of the microscopy optics unit and thus record an image of the object arranged in the object plane.

Of the at least two light sources, the (at least) first light source is configured to generate a first light and direct it toward the object region, the first light reflecting the fluorescent dye present in the object. Suitable to excite. For this purpose, the first light comprises light having a wavelength within the absorption range of the fluorescent dye. For PpIX, the first light can include wavelengths in the range of 380 nm to 420 nm.

Of the at least two light sources, the (at least) second light source is configured to generate a second light and direct it toward the object region, the second light visualizing a non-fluorescent region of the object. To help. For this purpose, the second light comprises light having a wavelength outside the absorption and emission range of the fluorescent dye.

One or more of the light sources may be narrow band. The narrow-band light source generates light according to the spectral intensity distribution, and the spectral intensity distribution has the maximum spectral emission intensity at the central wavelength, and the spectral emission intensity is 1% of the maximum spectral emission intensity. The difference between the long upper wavelength and the lower wavelength whose spectral emission intensity is 1% of the maximum spectral emission intensity and shorter than the central wavelength is 150 nm at the maximum, 100 nm at the maximum, more particularly 50 nm at the maximum, or at the maximum. 20 nm, or 10 nm at maximum.

One or more of the light sources may be individually controlled by the controller. That is, the light source can be controlled independently of other light sources. The intensity of the light generated by the individually controllable light sources can therefore be set individually. The ratio of the intensities of the light produced by the different light sources can therefore be varied. For example, the controller is configured to change the operating current and/or the operating voltage of the individually controllable light source, thereby changing the intensity of the light produced by the light source. The light source is, for example, a light emitting diode.

The (wavelength dependent) transmissivity of an observation filter is usually defined as the ratio of the intensity of light of one wavelength transmitted by the observation filter to the intensity of light of the same wavelength directed to the observation filter.

The transmission of the viewing filter is such that it blocks (does not transmit) the light used to excite the fluorescent dye, and transmits to a certain extent the light used to visualize the non-fluorescent regions. , And it is implemented in the best way to transmit fluorescence.

By being able to individually set the spectral intensity component of the light source, and thus of the light directed at the object, and by appropriately choosing the transmittance in the wavelength range used to visualize the non-fluorescent region, the following advantages are provided: can get.

First, the fluorescent and non-fluorescent regions can be perceived as nearly equal in brightness due to the intensity of the light generated by setting the individual light sources accordingly. The first light source can be used (substantially independent of the second light source) to set the intensity of the fluorescence emitted from the fluorescent region. The second light source can be used (substantially independent of the first light source) to set the intensity of the light reflected from the non-fluorescent region. The intensity of the light emitted from the fluorescent and non-fluorescent regions can thus be adjusted relative to each other, so that both regions appear to be of approximately equal brightness.

Moreover, all light sources can now be operated in their emission stable operating range. The emission spectrum of a light source is generally not stable at very low operating currents and/or voltages. That is, the emission spectrum changes as the operating current changes. To avoid this, all light sources can be operated in their emission stable operating range, for example by operating all light sources at at least 0.1% of their respective maximum power consumption. .. Used to visualize the non-fluorescent region so that the intensity of the light operated by the light source at at least 0.1% of its maximum power consumption is approximately equal to the intensity of the fluorescent light (ie the fluorescent and non-fluorescent regions are the same). In order to make it appear bright), the transmission of the observation filter in the wavelength range used for visualizing the non-fluorescent region can be correspondingly selected small. As a result, all light sources can operate in their stable emission operating range.

Furthermore, no illumination filter is needed.

The use of at least two light sources and at least two separate passbands of the observation filter for the visualization of the non-fluorescent region results in another advantage. The color rendering of the non-fluorescent area is the intensity of the light produced by the light source used to visualize the non-fluorescent area and the corresponding passband transmission provided for this purpose in the viewing filter. Can be set by appropriately selecting. In particular, the color rendering may be set to true color rendering.

In the embodiments described later, the transmittance of λ1 to λ2 of the observation filter is less than W1, is greater than W2 at λ3 to λ4, and greater than W3 at λ5 to λ6, and is λ1, λ2, λ3, λ4, λ5, λ6. Is a wavelength to which 350 nm<λ1<λ2≦λ3<λ4≦λ5<λ6<750 nm is applied, W1, W2 and W3 are values to which 0<W1<W2<W3<1 is applied, and the first light source is , Configured to generate a first light having a wavelength of λ1 to λ2, and the second light source configured to generate a second light having a wavelength of λ3 to λ4.

To simultaneously observe the fluorescent and non-fluorescent regions of an object mixed with PpIX, the viewing filter and the light source can be configured as follows:
350 nm<λ1<400 nm and/or 410 nm<λ2<440 nm, and/or 440 nm<λ3<460 nm and/or 480 nm<λ4<620 nm, and/or 530 nm<λ5<620 nm and/or 650 nm<λ6<750 nm, and/or Or W1=0.1% or W1=0.01%, and/or W2=1%, and/or W3=90%.

First Embodiment In order to observe the non-fluorescent region of the blue wavelength range, the following settings also apply, as is customary and desired in a particular application:
480 nm<λ4<520 nm, and/or the transmittance of λ3 to λ4 of the observation filter is greater than 10% and less than 60%, and/or the transmission of λ2+Δ to λ3-Δ and λ4+Δ to λ5-Δ of the observation filter. The ratio is less than 0.1×W1, 3 nm≦λ≦10 nm applies, and/or the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity, and/or The intensity of the second light below λ2 and above λ5 is at most 1% of its maximum spectral intensity.

In order to be able to set the color rendering of the non-fluorescent region to a particularly true color rendering, the microscopy system is arranged to generate a third light having a wavelength of λ7 to λ8 and direct it towards the object plane. Further included a third light source, where λ7, λ8 are wavelengths, and λ2<λ7<λ8<λ5. Some configurations used to achieve true color rendering of non-fluorescent regions by proper operation of the light source are described below.

Second Embodiment The transmittance of λ3 to λ6 of the observation filter is larger than W3. Advantageously, the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ2 and above λ5 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light below λ2 and above λ5 is at most 1% of its maximum spectral intensity.

Third Embodiment The observation filter has a transmittance of λ3 to λ4 of less than 5%, and 600 nm<λ4<λ5<620 nm applies. Advantageously, the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ2 and above λ5 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light below λ2 and above λ5 is at most 1% of its maximum spectral intensity.

Fourth Embodiment Still more preferably, the microscopy system according to the third embodiment further comprises an illumination filter which can be arranged between the light source and the object plane, the illumination filters of λ1 to λ3′ and λ4′ to λ4. The transmittance is greater than W3 and less than W1 at λ3′+Δ to λ4′−Δ and λ4+Δ˜, 3 nm≦Δ≦10 nm applies, and λ3′ and λ4′ are wavelengths and λ3<λ3′. <λ4′<λ4, 480 nm<λ3′<520 nm, and 520 nm<λ4′<550 nm, and λ4′−λ3′>20 nm apply. Advantageously, the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ2 and above λ4' is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light in λ4′ to λ4 is significantly higher and is at most 1% of its maximum spectral intensity below λ3′ and above λ5.

Fifth Embodiment The transmittance of λ3 to λ4 of the observation filter is less than 5%, the transmittance of λ7 to λ8 of the observation filter is larger than W2 and less than 5%, and λ4+Δ to λ7 of the observation filter. The transmittance of −Δ is less than W1, 3 nm≦Δ≦10 nm is applied, λ7 and λ8 are wavelengths, and λ4<λ7<λ8<λ5, and 480 nm<λ4<520 nm, and 520 nm<λ7<550 nm. , And λ7−λ4>20 nm, and λ5−λ8<30 nm. Advantageously, the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ2 and above λ7 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light below λ4 and above λ5 is at most 1% of its maximum spectral intensity.

Sixth Embodiment The transmittance of λ3 to λ4 of the observation filter is less than 5%, the transmittance of λ4+Δ to λ5-Δ of the observation filter is less than W1, and 480 nm<λ4<520 nmm and 530 nm<λ5. <560 nm and λ5-λ4>20 nm apply. The microscopy system further includes an illumination filter that can be arranged between the light source and the object plane, wherein the illumination filter has a transmittance of λ1 to λ4 greater than W3 and a transmittance of the illumination filter of λ4+Δ to λ5′−Δ. , W1 and the transmittance of λ5′ to λ6′ of the illumination filter is greater than W2 and less than 5%, and the transmittance of λ6′ to λ6 of the illumination filter is less than W1 and 3 nm≦Δ≦ 10 nm applies, λ5′, λ6′ are wavelengths, and λ4<λ5′<λ6′<λ6 applies, 530 nm<λ5′<570 nm and 600 nm<λ6′<620 nm apply. Advantageously, the intensity of the first light above λ3 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ2 and above λ5 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light below λ4 and above λ5 is at most 1% of its maximum spectral intensity.

For simultaneously observing the fluorescent and non-fluorescent regions of an object mixed with PpIX, a light source configured as follows is particularly suitable for the embodiments described above: The first light source is in the range 400 nm to 420 nm. It may be a light emitting diode having a spectral emission maximum and a full width at half maximum of 10 nm to 20 nm. The second light source may be a light emitting diode having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. The third light source can be a light emitting diode having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm.

Seventh Embodiment In order to simultaneously observe the fluorescent and non-fluorescent regions of an object mixed with fluorescein, the microscopy system produces a third light having a wavelength of λ5′ to λ6′ and directs it to the object. Further comprising a third light source configured to face the surface, an illumination filter positionable between the light source and the object plane, wherein the illumination filters have transmittances λ1 to λ4 greater than W3 and λ5′ of the illumination filter. ~λ6' is greater than W2 and less than 5%, λ5', λ6' are wavelengths, and λ5 <λ5'<λ6' applies, and the transmittance of λ3 to λ4 of the observation filter is It is less than 5%.

Advantageously, the transmittance of the illumination filter from λ4+Δ to λ5′−Δ is less than W1, and 3 nm≦Δ≦10 nm applies. Advantageously, the transmittance of the illumination filter between λ6' and 750 nm is less than W1. Advantageously, the transmission of the observation filter from λ4+Δ to λ5-Δ is less than W1, and 3 nm≦Δ≦10 nm applies.

The viewing filter and the light source can be configured as follows to simultaneously view the fluorescent and non-fluorescent regions of the object mixed with fluorescein:
350 nm<λ1<460 nm and/or 460 nm<λ2<480 nm, and/or 470 nm<λ3<490 nm and/or 490 nm<λ4<510 nm, and/or 520 nm<λ5<550 nm and/or 680 nm<λ6<750 nm, and/or Or 620 nm<λ5′<650 nm and/or 680 nm<λ6′<750 nm, and/or W1=0.1% or W1=0.01%, and/or W2=0.5%, and/or W3=90 %.

Advantageously, the intensity of the first light above λ5 is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the second light below λ3 and above λ5' is at most 1% of its maximum spectral intensity. Advantageously, the intensity of the third light below λ4 is at most 1% of its maximum spectral intensity.

For simultaneously observing the fluorescent and non-fluorescent regions of an object mixed with fluorescein, a light source configured as follows is particularly suitable for the embodiments described above: the first light source is in the range 440 nm to 470 nm. And a full width at half maximum in the range of 10 nm to 20 nm. The second light source can be a light emitting diode having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. The third light source may be a light emitting diode having a spectral emission maximum in the range of 600 nm to 640 nm and a full width at half maximum in the range of 10 nm to 20 nm.

Advantageously, the wavelength-dependent transmissivity of the illumination filter and the observation filter are adjusted to each other so that between 350 nm and 750 nm the absolute value of the wavelength-dependent transmissivity gradient of the illumination filter and the absolute value of the wavelength-dependent transmissivity gradient of the observation filter are adjusted. It is ensured that there are no wavelengths whose values and both are greater than 2%/nm. This prevents significant changes in the transmittance of the illumination and observation filters at a certain wavelength. Therefore, manufacturing tolerances on the transmittance of the illumination and viewing filters do not significantly affect the color accuracy and brightness of the different spectral ranges realized by the illumination and viewing filters.

Second Aspect The second aspect of the present invention relates to a method of operating a microscopy system, in particular one of the microscopy systems described herein.

が当てはまるように生成され、式中、

は、ＣＩＥ １９３１標準表色系のＣＩＥ標準色度図における白色点の色位置を表し、及び

は、ＣＩＥ １９３１標準表色系のＣＩＥ標準色度図における座標ｘ_ｆ，ｙ_ｆを有する色位置を表す。 The method includes generating illumination light and directing the generated illumination light at an object, generating an observation beam path that images the object at an image plane, and an observation filter is disposed in the observation beam path. It The illumination light is

Is generated so that

Represents the color position of the white point in the CIE standard chromaticity diagram of the CIE 1931 standard color system, and

Represents a color position having coordinates x _f and y _f in the CIE standard chromaticity diagram of the CIE 1931 standard color system.

と、ＣＩＥ １９３１標準表色系のＣＩＥ標準色度図における座標ｘ_Ｂ及びｙ_Ｂを有する色位置

との間の距離│・│は、

として定義される。 Color position having coordinates x _A and y _A in the CIE standard chromaticity diagram of the CIE 1931 standard color system

And a color position having coordinates x _B and y _B in the CIE standard chromaticity diagram of the CIE 1931 standard color system.

The distance between

Is defined as

の座標ｘ_ｆ，ｙ_ｆは、

によって定義され、式中、Ｘ、Ｙ、Ｚは、

によって定義されるＣＩＥ １９３１標準表色系の三刺激値を表し、
式中、Ｉ（λ）は、照明光の強度を表し、Ｔ（λ）は、観察フィルタ（２３）の透過率（４１、６１、８１、１０１、１２１、１４１、１６１）を表し、

は、ＴＲＧ９０９９２６ ＣＩＥ １９３１標準表色系のスペクトル値関数を表し、及びｋは、定数である。 Color position

The coordinates x _f , y _f of

Where X, Y and Z are

Represents the tristimulus values of the CIE 1931 standard color system defined by
In the formula, I(λ) represents the intensity of the illumination light, T(λ) represents the transmittance (41, 61, 81, 101, 121, 141, 161) of the observation filter (23),

Represents the spectral value function of the TRG 909926 CIE 1931 standard color system, and k is a constant.

The white point can be, for example, the white point D50 having the coordinates x=0.3457 and y=0.3585 in the CIE standard chromaticity diagram of the CIE 1931 standard color system. Alternatively, the white point may be, for example, the white point D65 having coordinates x=0.127 and y=0.329 in the CIE standard chromaticity diagram of the CIE 1931 standard color system. As yet another alternative, the white point is at a distance of at most 0.2, especially at most 0.1 or at most 0.05 from the color position of the white point D50 in the CIE 1976 u'v' chromaticity diagram. It may correspond to a color value having a color position.

と、ＣＩＥ １９７６ ｕ’ｖ’色度図における座標ｕ’_Ｂ及びｖ’_Ｂを有する色位置

との間の距離│・│は、

として定義される。 Color position with _A and v _'A' coordinates u in the chromaticity diagram 'CIE 1976 u'v

When the color position with _B and v _'B' coordinate u in the chromaticity diagram 'CIE 1976 u'v

The distance between

Is defined as

又は

が当てはまるように生成される。波長λにわたる積分は、３８０ｎｍ〜７８０ｎｍで積分される。 Preferably, the illumination light is

Or

Is generated so that Integration over wavelength λ is integrated from 380 nm to 780 nm.

は、強度Ｉ（λ）の照明光が白い物体に衝突し、その白い物体から出た光が透過率Ｔ（λ）の観察フィルタによりフィルタ処理された場合に得られる、ＣＩＥ標準表色系のＣＩＥ標準色度図における色位置を表す。ＣＩＥ １９３１標準色度図における白色点からの要求される小さい距離は、白い物体がほぼ白として観察できることを意味する。一般的な物体に当てはめた場合、これは、その物体がほぼトゥルーカラーとして観察できることを意味する。これにより、外科医にとって術野の方位特定が容易となる。 Expression (1) represents the condition of the maximum color value distance in the CIE 1931 standard chromaticity diagram. in this case,

Of the CIE standard color system, which is obtained when illumination light of intensity I(λ) strikes a white object and the light emitted from the white object is filtered by an observation filter of transmittance T(λ). The color position in the CIE standard chromaticity diagram is shown. The required small distance from the white point in the CIE 1931 standard chromaticity diagram means that white objects can be seen as nearly white. When applied to a general object, this means that the object can be viewed as nearly true color. This makes it easier for the surgeon to identify the orientation of the surgical field.

Illumination light is generated by, for example, a plurality of light sources that generate light in different wavelength ranges. The step of generating illumination light may include the step of setting, in particular changing, the energy supplied to at least one of the plurality of light sources for generating the illumination light.

The method may be performed by the microscopy system described herein.

Third Aspect The third aspect of the present invention relates to a microscopy method in which a fluorescent region and a non-fluorescent region can be recognized as almost equal brightness.

For this purpose, the microscopy method comprises the steps of generating illumination light and directing the generated illumination light at an object, generating an observation beam path for imaging the object on an image plane, the observation filter comprising: A first value, which is arranged in the observation beam path and represents an average value of the transmittance of the observation filter over the first wavelength range, and a second value, which represents an average value of the transmittance of the observation filter over the second wavelength range. Is less than the second value, and the second value is less than the third value, which represents the average value of the transmittance of the observation filter over the third wavelength range, and represents the average value of the intensity of the illumination light over the first wavelength range. The fourth value is greater than a fifth value that represents an average value of the intensity of the illumination light over the second wavelength range, and the fifth value represents a first value that represents the average value of the intensity of the illumination light over the third wavelength range. Greater than six values, the first, second and third wavelength ranges do not overlap each other and in each case are 350 nm to 1000 nm.

The first wavelength range corresponds to the wavelength range in which the fluorescent dye present in the object has a high absorption. In order to be able to efficiently excite the fluorescent dye, the illumination light in the first wavelength range has a high intensity characterized by a fourth value. In order that the light reflected by the object in the first wavelength range does not substantially overwhelm the fluorescence generated only in the third wavelength range, the observation filter is It has a low transmission characterized by a value of.

In order to be able to observe the fluorescence efficiently, the observation filter has a high transmittance in the third wavelength range, characterized by a third value. The illumination light in the third wavelength range has a low intensity, characterized by a sixth value, so that the fluorescence light is not overwhelmed by the illumination light in the third wavelength range reflected by the object.

The second wavelength range serves to visualize the non-fluorescent areas of the object. For this purpose, the illumination light has a medium intensity in the second wavelength range, said intensity being characterized by a fifth value, the observation filter being It has an average transmission characterized by a value.

Light in the first and second wavelength ranges transmitted by the viewing filter results in visualization of the non-fluorescent regions of the object and thus contributes mainly to the brightness of the non-fluorescent regions in the image plane. Light in the third wavelength range transmitted by the viewing filter results in visualization of the fluorescent region of the object and thus contributes mainly to the brightness of the fluorescent region in the image plane. The above relationship of the first to sixth values with respect to one another has the effect that the non-fluorescent and fluorescent regions of the object appear to be of approximately equal brightness. In addition, they are coordinated with each other to allow for near-true color viewing of non-fluorescent regions.

The first wavelength range may include wavelengths for which the absorptivity of the fluorescent dye present in the object-normalized to its maximum-is at least 5%, in particular at least 10%, more particularly at least 50%. .. Therefore, the first wavelength range includes wavelengths that can sufficiently excite the fluorescent dye.

The third wavelength range may include wavelengths where the emissivity of the fluorescent dye present in the object-normalized to its maximum-is at least 5%, in particular at least 10%, more particularly at least 50%. .. Therefore, the third wavelength range includes wavelengths that can sufficiently excite the fluorescent dye.

The second wavelength range is a maximum of 20%, in particular a maximum of 10%, more in particular a maximum of 5% of the absorption of the fluorescent dye present in the object-normalized to its maximum-and the object It is possible to include only those wavelengths whose emission rate of the fluorescent dye present therein-normalized to its maximum-is at most 20%, in particular at most 10%, and more particularly at most 5%. Thus, the second wavelength range comprises substantially only those wavelengths that are outside the wavelength range in which the fluorescent dye can be efficiently excited and produce fluorescence.

Due to the use of the fluorescent dye protoporphyrin IX, the first wavelength range preferably comprises a wavelength of 405 nm and/or a wavelength of 390 nm to 420 nm, the second wavelength range preferably a wavelength of 450 nm to 600 nm. And the third wavelength range preferably includes wavelengths from 620 nm to 720 nm.

Due to the use of the fluorescent dye fluorescein, the first wavelength range preferably comprises wavelengths from 480 nm to 500 nm, the second wavelength range preferably comprises wavelengths from 620 nm to 750 nm and the third wavelength range. Preferably comprises a wavelength of 550 nm to 600 nm.

Preferably, the ratio between the first value and the second value is at most 1/100, especially at most 1/1000. Preferably, the ratio of the second value to the third value is at most 0.9, especially at most 0.8, more particularly at most 0.5. Preferably, the ratio of the fourth value to the fifth value is at least 2, especially at least 5. Preferably, the ratio of the fourth value to the sixth value is at least 1000, especially at least 10,000. As a result, both near equal brightness and near true color rendering are achieved.

Illumination light can be generated using multiple (narrow band) light sources, for example using the light sources described in the other aspects. At least one of the light sources produces light having a wavelength in the first wavelength range and at least one of the light sources produces light having a wavelength in the second wavelength range. At least one of the light sources may be individually controllable to provide a ratio of the intensity of light having a wavelength in the first wavelength range to the intensity of light having a wavelength in the second wavelength range. Can be changed.

Preferably, the light source produces substantially no light having a wavelength within the third wavelength range. This prevents the light generated by the light source from disturbing the fluorescence within the third wavelength range.

An alternative microscopy method includes generating illumination light and directing the generated illumination light at an object, generating an observation beam path that images the object at an image plane, the illumination light comprising: Ratio of the second feature value to the second feature value is in the range of 20/1 to 1/20, and the first feature value is the observation beam path at the first position in the image plane. Is the value of the characteristic variable relating to the light intensity of, in its first position, the color value of the light in the observation beam path is within the first color value range of the color space and the second characteristic value is The value of the characteristic variable for the intensity of the light in the observation beam path at the second position in the image plane, where the color value of the light in the observation beam path is the second color value in the color space. Within range.

Preferably, the illumination light has a ratio of the first characteristic value to the second characteristic value within the range of 10/1 to 1/10, or 5/1 to 1/5, or 3/1 to 1/3. Is generated to have the value of.

A projected image of the object is created in the image plane through the observation beam path. When the fluorescent dye present in the object is excited by the illumination light, the projected image generally includes a fluorescent region (first position) and a non-fluorescent region (second position). The intense fluorescence region (first position) has a color value typical of fluorescent dyes in the projected image, which is within the first color value range. The weakly fluorescent and non-fluorescent areas (second position) have other color values in the projected image, which are within the second color value range. Therefore, the first and second color value ranges are ranges in which the same color space does not overlap.

The fluorescent region (first position) of the object in the image plane can be distinguished from the non-fluorescent region (second position) based on the color values typical of fluorescent dyes. In order for an observer or controller to be able to distinguish between both fluorescent and non-fluorescent regions, the fluorescent and non-fluorescent regions should appear to be of approximately equal brightness, i.e. approximately equal intensity or approximately equal intensity in the image plane. It is advantageous to have a luminous flux of Luminous flux is the ratio of radiant power visible to the human eye and is weighted by the visibility curve of the human eye.

This is realized in the microscopy method by generating the illumination light so that the ratio between the first feature value and the second feature value has a value within the range of 20/1 to 1/20. It The first feature value is, for example, the intensity of the light in the observation beam path at a first position in the image plane, i.e. in the image plane, where the light in the observation beam path has a color value typical of fluorescent dyes. Represents the average value of. In this example, the second feature value is the observation beam path at a second position in the image plane, i.e. at a position in the image plane where the light in the observation beam path does not have a color value typical of fluorochromes. Represents the average value of the light intensity inside.

The first characteristic value and the second characteristic value represent the values of characteristic variables of the same type, respectively. For example, when the first feature value represents the maximum value, the second feature value also represents the maximum value. Alternatively, if the first feature value represents, for example, an average value, the second feature value also represents the average value.

The first characteristic value can represent a maximum value or an average value of the light intensity (or luminous flux) of the observation beam path at the first position. The second feature value can represent a maximum or average value of the light intensity (or luminous flux) of the observation beam path at the second position.

The microscopy method is a step of identifying a first position in the image plane, in which the color value of the light in the observation beam path is within a first color value range in the color space. A step and a step of identifying a second position in the image plane at which the color value of the light in the observation beam path lies within a second range of color values in the color space. A step of illuminating light according to the intensity of the light of the observation beam path at the specified first position and the intensity of light of the observation beam path at the specified second position. Is generated according to. As a result, the first and second positions in the image plane are identified, and the illumination light is generated according to the intensity at these positions.

The microscopy method may further include the step of identifying the light intensity of the observation beam path at the first and second positions to identify the luminous flux of the light of the observation beam path at the first and second positions. .. For example, an image detector is arranged in the image plane and is arranged to record a color image of the projected image, on the basis of which, firstly, the first and second positions can be distinguished and secondly, The light intensity of the observation beam path at the first and second positions can be specified.

The microscopy method is a step of recording an image of an object in an image plane, wherein the image has a plurality of image points, and the color value at the image points of the image is set to a first color value range and/or A step of comparing with a second color value range, and a step of identifying a first image point from a plurality of image points based on the result of the comparison, the first image point is the first image point in the image plane. The color value at the first image point corresponding to the position is within the first color value range of the color space, and the second image point is identified from the plurality of image points based on the result of the step and comparison. The second image point corresponds to a second position in the image plane, and the color value at the second image point is within the second color value range of the color space. It can further be included.

The microscopy method includes the step of identifying a first feature value based on the identified intensity of light in the observation beam path at the identified first position, and the light in the observation beam path at the identified second position. Determining the second feature value based on the identified intensity of the illumination light, the illumination light being responsive to the identified first feature value, and the identified second feature value. Is generated according to. Thus, the first and second characteristic values are based in particular on the intensity values identified or measured at the first and second positions previously identified on the recorded image, and in particular on the first and second Specific, eg calculated, based on those color values by comparison of the second range of color values. The first and second feature values thus obtained are then used to generate illumination light.

Illumination light is generated by, for example, a plurality of light sources that generate light in different wavelength ranges. The step of generating illumination light may include the step of setting, in particular changing, the energy supplied to at least one of the plurality of light sources to generate the illumination light in response to the first and second characteristic values. it can.

In particular, the illumination light can be generated irrespective of the intensity of the light of the observation beam path at the third position in the image plane, and the color value of the light of the observation beam path at the third position is It is outside the first and second color value range. That is, only the light intensity of the observation beam path at the first and second positions is used to generate the illumination light. The light intensity of the observation beam path at other locations in the image plane is not used to generate the illumination light.

The first color value range and the second color value range are such that the minimum color difference between the first color value range and the second color value range in the CIE 1976 u'v' chromaticity diagram is at least 0. It may be selected to be 01, especially at least 0.1.

Furthermore, the first color value range is limited to color values having a color difference of at most 0.1, in particular at most 0.03 or at most 0.01 with respect to the reference color value in the CIE 1976 u'v' chromaticity diagram. The reference color value is at least 5%, in particular at least 10%, more particularly at least 50% of the emissivity of the fluorescent dye present in the object-normalized to its maximum value- It corresponds to light within the reference wavelength range that includes only wavelengths. The reference color value is, for example, a color value corresponding to the emission spectrum of the fluorescent dye in the object. Therefore, only color values whose degree of separation from the reference color values does not exceed the above-mentioned maximum difference belong to the first color value range.

When the fluorescent dye is PpIX, the first color value range preferably includes a color value corresponding to light having a wavelength of 635 nm. Furthermore, the first color value range is at most 0.1, in particular at most 0.03 or at most 0. 0 with respect to a reference color value corresponding to light having a wavelength of 635 nm in the CIE 1976 u'v' chromaticity diagram. It is preferable to be limited to color values having a color difference of 01.

When the fluorescent dye is fluorescein, the first color value range preferably includes a color value corresponding to light having a wavelength of 530 nm. Furthermore, the first color value range is at most 0.1, in particular at most 0.03 or at most 0,0 with respect to a reference color value corresponding to light having a wavelength of 530 nm in the CIE 1976 u'v' chromaticity diagram. It is preferable to be limited to color values having a color difference of 01.

The features of the first, second and third aspects can be combined with each other.

The observation filters and illumination filters described herein can be embodied as interference filters, for example. Interference filters include multiple layers of materials having different optical properties, such as different indices of refraction. Each layer can have a different thickness. The number of layers can be 2,300 to hundreds, depending on the requirements for the filter. The transmittances described herein can be realized by the specific choice of thickness and optical properties of each layer.

Aids are known for identifying the layer thickness and optical properties of filters according to a given wavelength-dependent transmission. The order of such layers is designed using a simulation program that uses as input data the optical properties of the materials used (refractive index and absorption, dispersion depending on wavelength) and the desired transmission and/or reflection spectra. it can. The simulation program also outputs simulated transmission and/or reflectance spectra, layer sequence and/or layer thickness and optical properties and/or materials used. The calculation can be performed iteratively. In this way, filters with complex requirements can also be designed and manufactured, for example multi-band filters.

Embodiments of the present invention will be described in more detail below with reference to the drawings.

1 shows a microscopy system according to the present invention. 3 shows absorption and emission spectra of PpIX and fluorescein. 3 shows illumination light spectra of the two light sources of the first embodiment. 5 shows the wavelength-dependent transmittance of the observation filter of the first embodiment. The illumination light spectrum of three light sources of 2nd-6th embodiment is shown. The wavelength-dependent transmittance of the observation filter of 2nd embodiment is shown. 6 shows a CIE 1931 standard chromaticity diagram for explaining the color values that can be realized for a white object according to the second embodiment. The wavelength-dependent transmittance of the observation filter of 3rd embodiment is shown. 7 shows a CIE 1931 standard chromaticity diagram for explaining the color values that can be realized for a white object according to the third embodiment. The wavelength-dependent transmittance of the observation filter of 4th Embodiment is shown. FIG. 11 shows a CIE 1931 standard chromaticity diagram for explaining color values that can be realized for a white object according to the fourth embodiment. The wavelength-dependent transmittance of the illumination filter and the observation filter of 5th Embodiment is shown. 9 shows a CIE 1931 standard chromaticity diagram for explaining color values that can be realized for a white object according to the fifth embodiment. The wavelength-dependent transmittance of the illumination filter and the observation filter of 6th Embodiment is shown. The illumination light spectrum of the three light sources of 7th embodiment is shown. The wavelength-dependent transmittance of the illumination filter and observation filter of the seventh embodiment is shown. FIG. 11 shows a CIE 1931 standard chromaticity diagram for explaining color values that can be realized for a white object according to the seventh embodiment. 1 illustrates one exemplary configuration of a microscopy system for matching the brightness of fluorescent and non-fluorescent object regions for protoporphyrin IX. 1 illustrates one exemplary configuration of a microscopy system for matching the brightness of fluorescent and non-fluorescent object regions for fluorescein. Figure 3 shows a schematic view of a projected image of an object in the image plane. 20 shows a schematic view of the image of the projected image shown in FIG. 20, the image consisting of a plurality of image points. 3 shows a CIE 1931 standard chromaticity diagram for explaining a color value range. 1 illustrates one exemplary microscopy method for matching the brightness of fluorescent and non-fluorescent object regions.

FIG. 1 illustrates one exemplary microscopy system 1 for viewing fluorescent and non-fluorescent regions of an object simultaneously.

The microscope inspection system 1 includes an illumination device 3. The illumination device 3 comprises a plurality of light sources 5, an illumination optical unit 7, and an illumination filter 9 which may optionally be arranged in the observation beam path 10 formed by the illumination optical unit 7. This is indicated by the double-headed arrow in FIG. The plurality of light sources 5 and the illumination filter 9 allow the illumination device 3 to generate various illumination light spectra and direct them to the object region 11.

The microscopy system 1 includes a controller 12 configured to control the plurality of light sources 5 individually, ie independently of each other. For example, the light source is two or more light emitting diodes. Each of the light emitting diodes can be individually controlled by the controller 12. For example, the controller 12 controls an operating current and/or an operating voltage supplied to generate light and/or applied to each light emitting diode, respectively. As a result, the intensity of the light generated by the light source 5 can be set individually.

The observation beam path 10 allows the illuminating device 3 to direct the illumination light to the object region 11 in which an object 13 can be arranged, which can be mixed with a fluorescent dye. The fluorescent dye supplied to the object 13 is very much accumulated in a certain area (fluorescent area) of the object 13. These areas include, for example, tumor cells, to which fluorescent dyes bind. The fluorescent dye is not accumulated in other regions (non-fluorescent regions) of the object 13 or is accumulated only slightly. These areas do not include, for example, tumor cells.

The microscopy system 1 further comprises a microscopy optics unit 14, which in this example comprises an objective lens 15 and further lenses 16 and 17. The microscopy optics unit 14 is configured to image the object region 11, in particular the object plane 18, on an image plane 19. In this example, the detection area of the photodetector 20 of the microscopy system 1 is arranged on the image plane 19. The photodetector 20 can be, for example, an image sensor. The photodetector 20 outputs a signal indicating the intensity of light that has collided with the detection area of the photodetector 20. The photodetector 20 is connected to the controller 12, which receives the signal 21 output thereby from the photodetector 20. The controller can process and display the images produced by the photodetector 20. Instead of or in addition to the photodetector 20, an eyepiece can be provided, which is suitable for imaging the object plane 18 with the microscopy optics unit 14 on the retina of the eye.

The observation filter 23 can be arranged in an observation beam path 22 provided by the microscopy optics unit 14, said observation beam path focusing the object plane 18 on an image plane 19. In addition, an actuator (not shown) can be provided, which can introduce the observation filter 23 into the observation beam path 22 and pull the observation filter 23 back out of the observation beam path 22. This is indicated by the double-headed arrow in FIG.

Depending on the configurations of the plurality of light sources 5, the optical filter 9 and the observation filter 23, the fluorescent and non-fluorescent regions of the object 13 can be observed simultaneously. Various embodiments of the fluorescent dyes protoporphyrin IX (PpIX) and fluorescein are described below.

FIG. 2 shows the wavelength-dependent absorption spectrum of _PpIX as graph A _PpIX , the wavelength-dependent emission spectrum of _PpIX as graph E _PpIX , the wavelength-dependent absorption spectrum of fluorescein as graph A _FL , and the wavelength-dependent emission spectrum of fluorescein. Shown as graph E _FL .

The absorption spectrum PpIX of the fluorescent dye PpIX from 350 nm to 430 nm has a normalized absorption intensity of more than 0.2. The normalized absorption intensity is normalized to the maximum absorption intensity, i.e. the normalized absorption spectrum has only values from 0 to 1. Therefore, the fluorescent dye PpIX can be efficiently excited in the range of 350 nm to 430 nm. The fluorescent dye PpIX has an absorption maximum at about 405 nm. The fluorescent dye PpIX fluoresces in the spectral range of about 600 nm to 750 nm, with the primary maximum of emission intensity at 635 nm and the secondary maximum at about 705 nm.

The fluorescent dye fluorescein has a normalized absorption intensity of greater than 0.2 at about 450 nm to 530 nm. Therefore, the fluorescent dye fluorescein is fully excitable in this range. The absorption spectrum of fluorescein has a maximum at about 495 nm. The fluorescent dye fluorescein emits light in the range of about 490 nm to 650 nm. The maximum of the emission spectrum is at about 520 nm.

Example of First Embodiment FIG. 3 shows illumination light spectra (spectral intensity distributions) 31, 32 of the first and second light sources according to the first embodiment. The first light source serves essentially only to excite the fluorescent dye PpIX, ie to visualize the fluorescent region of the object. The second light source serves essentially only to visualize the non-fluorescent areas of the object.

The first light source produces an illumination light spectrum 31 having a spectral emission maximum in the range 400 nm to 410 nm and a full width at half maximum in the range 10 nm to 20 nm. Below about 370 nm and above about 450 nm, the intensity of the first light produced by the first light source is at most 1% of its maximum spectral intensity. The first light source is, for example, a light emitting diode.

The second light source produces an illumination light spectrum 32 having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. Below about 440 nm and above about 500 nm, the intensity of the second light produced by the second light source is at most 1% of its maximum spectral intensity. The second light source is, for example, a light emitting diode.

The first and second light sources operate such that the maximum spectral intensity of the first light source is at most 20 times and at least 3 times the magnitude of the maximum spectral intensity of the second light source.

FIG. 4 shows the wavelength-dependent transmittance 41 of the observation filter 23 according to the first embodiment. At λ1=360 nm to λ2=430 nm, the transmittance 41 is about 0.01%. At λ3=435 nm to λ4=500 nm, the transmittance 41 is about 55%. At λ5=600 nm to λ6=750 nm, the transmittance 41 is about 95%. In the remaining wavelength range of λ1 to λ6, the transmittance 41 is 0.001% at maximum. Furthermore, W1=0.1%, W2=1% and W3=80% apply.

Therefore, the observation filter 23 suppresses the first light reflected by the object 13, and the first light has a high intensity substantially only at λ1 and λ2. Furthermore, the second light reflected by the object 13 (which second light has a high intensity substantially only at λ3 to λ4) is transmitted by the observation filter 23, so that the non-fluorescent light of the object 13 is emitted. The area can be observed as bluish. Furthermore, the fluorescence emitted from the object 13 (the fluorescence having substantially high intensity only at λ5 to λ6) is transmitted by the observation filter 23, so that the fluorescent region of the object 13 has a reddish color. It can be observed as a value.

The ability to set the intensity of the first and second light separately (see FIG. 3) and the specific choice of the transmission 41 of the observation filter 23 provides two main advantages. First, the fluorescent region and the non-fluorescent region can be recognized as almost equal brightness by setting the intensity of the light source accordingly. Secondly, due to the specific choice of the transmission 41 of λ3 to λ4 and λ5 to λ6, the light sources can now operate in their stable emission range. Furthermore, no illumination filter is needed.

Example of Second to Sixth Embodiments FIG. 5 shows illumination light spectra (spectral intensity distributions) 51, 52 and 53 of the first, second and third light sources according to the second to sixth embodiments. The first light source serves essentially only to excite the fluorescent dye PpIX, ie to visualize the fluorescent region of the object. The second and third light sources serve substantially only to visualize the non-fluorescent areas of the object.

The first and second light sources correspond to those of the example for the first embodiment. That is, the illumination light spectrum 31 of the first light source of the first embodiment shown in FIG. 3 and the illumination light spectrum 51 of the first light source of the second to sixth embodiments shown in FIG. Same as above, the illumination light spectrum 32 of the second light source of the first embodiment shown in FIG. 3 and the illumination light spectrum 52 of the second light source of the second to sixth embodiments shown in FIG. Are the same.

The third light source produces an illumination light spectrum 53 having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. Below about 470 nm and above about 630 nm, the intensity of the third light produced by the third light source is at most 1% of its maximum spectral intensity. The third light source is, for example, a light emitting diode.

The first and third light sources operate such that the maximum spectral intensity of the first light source is at most 20 times and at least 3 times the magnitude of the maximum spectral intensity of the third light source.

Example of Second Embodiment FIG. 6 shows the wavelength-dependent transmittance 61 of the observation filter 23 according to the second embodiment. At λ1=350 nm to λ2=435 nm, the transmittance 61 is about 0.005%. At λ3=440 nm to λ4=λ5=600 nm and λ5 to λ6=750 nm, the transmittance 61 is about 95%. Furthermore, W1=0.01%, W2=1% and W3=80% apply.

Therefore, the observation filter 23 suppresses the first light reflected by the object 13, and the first light has a high intensity substantially only at λ1 and λ2. Furthermore, the second and third light reflected by the object 13 (the second and third light having a high intensity substantially only at λ3 to λ4) are transmitted by the observation filter 23, so that The non-fluorescent region of the object 13 can be observed in almost true color. Furthermore, the fluorescence emitted from the object 13 (the fluorescence having substantially high intensity only at λ5 to λ6) is transmitted by the observation filter 23, so that the fluorescent region of the object 13 has a reddish color. It can be observed as a value.

The fact that the first, second and third light intensities can be set individually (see FIG. 5) and the specific selection of the transmission 61 of the observation filter 23 has three main advantages. To be First, the fluorescent region and the non-fluorescent region can be recognized as almost equal brightness by setting the intensity of the light source accordingly. Furthermore, by the specific choice of the transmission 61 of λ3 to λ4 and λ5 to λ6, the light sources can now operate in their stable emission range. Finally, by being able to set the second and third light intensities separately, the color rendering of the non-fluorescent regions can be set so that the non-fluorescent regions can be viewed in near true color. Moreover, no illumination filter is needed.

FIG. 7 shows a CIE 1931 xy standard chromaticity diagram including a spectrum locus S and a pure purple locus P. White circles represent the D50 white point where x=0.3457 and y=0.3585. The x-marks of approximately x=0.26 and y=0.45 represent the color values by which a normal observer recognizes a white object using the microscopy system according to the second embodiment, and the observation filter is shown in FIG. And the maximum spectral intensity of the light produced by the first light source is in each case the magnitude of the maximum spectral intensity of the light produced by the second and third light sources, respectively. It is about 20 times that. Therefore, the non-fluorescent region of the object can be recognized as almost true color. In addition, the result of this configuration is that the fluorescent and non-fluorescent areas of the object are of approximately equal brightness.

Example of Third Embodiment FIG. 8 shows a wavelength-dependent transmittance 81 of the observation filter 23 according to the third embodiment. At λ1=350 nm to λ2=435 nm, the transmittance 81 is about 0.005%. At λ3=440 nm to λ4=610 nm, the transmittance 81 is about 2%. At λ5=620 nm to λ6=750 nm, the transmittance 81 is about 95%. Furthermore, W1=0.01%, W2=1% and W3=80% apply.

Therefore, the observation filter 23 suppresses the first light reflected by the object 13, and the first light has a high intensity substantially only at λ1 and λ2. Furthermore, the second and third light reflected by the object 13 (the second and third light having a high intensity substantially only at λ3 to λ4) are transmitted by the observation filter 23, so that , The non-fluorescent region of the object 13 can be observed as an almost true color. Furthermore, the fluorescence emitted from the object 13 (the fluorescence having substantially high intensity only at λ5 to λ6) is transmitted by the observation filter 23, so that the fluorescent region of the object 13 has a reddish color. It can be observed as a value.

The advantage of the example of the second embodiment is obtained by the fact that the intensity of the first, second and third light can be set individually (see FIG. 5), λ3 compared to the second embodiment. Due to the low transmittance 81 at λ4, a better accuracy of observation of the non-fluorescent region is realized. In addition, the second and third light sources can operate at higher power compared to the second embodiment, which thus improves the stability of the generated illumination light spectrum 52,53. Furthermore, no illumination filter is needed.

FIG. 9 shows a CIE 1931 xy standard chromaticity diagram including a spectrum locus S and a pure purple locus P. White circles represent the D50 white point where x=0.3457 and y=0.3585. The x-marks of approximately x=0.33 and y=0.38 represent the color values by which a normal observer perceives a white object using the microscopy system according to the third embodiment, and the observation filter is shown in FIG. Configured such that the maximum spectral intensity of the light produced by the first light source is in each case about 7 times the magnitude of the maximum spectral intensity of the light produced by the second light source. And the maximum spectral intensity of the light produced by the first light source is about 5 times the magnitude of the maximum spectral intensity of the light produced by the third light source. Therefore, the non-fluorescent region of the object can be recognized as almost true color. In addition, the result of this configuration is that the fluorescent and non-fluorescent areas of the object are of approximately equal brightness.

Example of Fourth Embodiment FIG. 10 shows the wavelength-dependent transmittance 101 (solid line) of the observation filter 23 and the wavelength-dependent transmittance 102 (broken line) of the illumination filter 9 according to the fourth embodiment. The example observation filter for the fourth embodiment corresponds to the example observation filter for the third embodiment (λ1=350 nm, λ2=435 nm, λ3=440 nm, λ4=610 nm, λ5=620 nm, λ6=750 nm). ..

At 360 nm to λ3′=500 nm, the transmittance 102 of the illumination filter 9 is about 95%. At approximately λ3′ to approximately λ4′=560 nm, the transmittance 102 of the illumination filter 9 is approximately 0.005%. From λ4' to approximately λ4, the transmittance 102 of the illumination filter 9 is about 95%. The transmittance 102 of the illumination filter 9 is approximately 0.005% at approximately λ4 to λ6. Furthermore, W1=0.01%, W2=1% and W3=80% apply.

The illumination filter and the observation filter jointly have substantially the same effect as the example observation filter for the fifth embodiment. The observation filter suppresses the first light reflected by the object 13, said first light having a high intensity substantially only at λ1 and λ2 and passing through the illumination filter without being substantially attenuated. To do. Further, the second light reflected by the object 13 is restricted by the illumination filter and the observation filter to a wavelength substantially included only in λ3 to λ3'. Further, the third light reflected by the object 13 is restricted by the illumination filter and the observation filter to a wavelength substantially included only in λ4′ to λ4. Light within the wavelength range of λ3' to λ4' is effectively suppressed by the illumination filter and the observation filter. Therefore, the non-fluorescent region of the object 13 can be observed as almost true color. Furthermore, the fluorescence emitted from the object 13 and having a high intensity substantially only at λ5 to λ6 is transmitted by the observation filter 23, so that the fluorescence region of the object 13 is observable with a reddish color value.

FIG. 11 shows a CIE 1931 xy standard chromaticity diagram including a spectrum locus S and a pure purple locus P. White circles represent the D50 white point where x=0.3457 and y=0.3585. The x-marks of approximately x=0.35 and y=0.36 represent the color values by which a normal observer perceives a white object using the microscopy system according to the fourth embodiment. Is configured as shown in FIG. 10, the maximum spectral intensity of the light generated by the first light source is, in each case, the magnitude of the maximum spectral intensity of the light generated by the second light source. Is about 10 times, and the maximum spectral intensity of the light generated by the first light source is about 5 times the magnitude of the maximum spectral intensity of the light generated by the third light source. Therefore, the non-fluorescent region of the object can be recognized as almost true color. In addition, the result of this configuration is that the fluorescent and non-fluorescent areas of the object are of approximately equal brightness.

The second light intensity 52 and the third light intensity 53 can be set individually (see FIG. 5), and the specifics of the transmittance 101 of the observation filter 23 and the transmittance 102 of the illumination filter 9 can be set specifically. This choice offers three main advantages. First, by appropriately setting the intensity of the light source, the fluorescent region and the non-fluorescent region can be recognized as having almost the same brightness. Furthermore, the light sources can now operate in their stable emission range, since the light produced by the second and third light sources is attenuated by the observation filter 23 and the illumination filter 9 to a sufficient extent. is there. Finally, as a result of being able to set the second and third light intensities separately, the color rendering of the non-fluorescent regions can be set such that the non-fluorescent regions are visible as nearly true color.

The broken line shown in FIG. 11 represents the color of the non-fluorescent region of the object obtained by the change in the ratio of the second light intensity 52 (maximum thereof) to the third light intensity 53 (maximum thereof). In this way, a white point different from D50 can also be set advantageously.

Example of Fifth Embodiment FIG. 12 shows the wavelength-dependent transmittance 121 of the observation filter 23 according to the fifth embodiment. At λ1=350 nm to λ2=453 nm, the transmittance 121 is about 0.005%. At λ3=440 nm to λ4=500 nm, the transmittance 121 is about 2%. The transmittance 121 is about 0.005% at about λ4 to about λ7. At λ7=535 nm to λ8=610 nm, the transmittance 121 is about 2%. At λ5=620 nm to λ6=750 nm, the transmittance 121 is about 95%. Furthermore, W1=0.01%, W2=1% and W3=80% apply.

Therefore, the observation filter 23 suppresses the first light reflected by the object 13, and the first light has a high intensity substantially only at λ1 and λ2. Further, the second light reflected by the object 13 (the second light having a high intensity substantially only at λ3 to λ4) is transmitted by the observation filter 23. Further, the third light reflected by the object 13 is efficiently transmitted by the observation filter 23 substantially only at λ7 to λ8. Therefore, the non-fluorescent region of the object 13 can be observed as almost true color. Furthermore, the fluorescence emitted from the object 13 (the fluorescence having substantially high intensity only at λ5 to λ6) is transmitted by the observation filter 23, so that the fluorescent region of the object 13 has a reddish color. It can be observed as a value.

An advantage of the example for the third embodiment is obtained by the fact that the first, second and third light intensities can be set individually (see FIG. 5), but with the third embodiment. By comparison, by more significantly spectrally separating the second and third light reflected at the object 13, the truecolor rendering of the non-fluorescent region may be much better configurable. In addition, the second and third light sources can operate at higher power compared to the example for the second embodiment, which therefore improves the stability of the light spectrum produced. Furthermore, no illumination filter is needed.

FIG. 13 shows a CIE 1931 xy standard chromaticity diagram including a spectrum locus S and a pure purple locus P. White circles represent the D50 white point where x=0.3457 and y=0.3585. The x-marks of approximately x=0.34 and y=0.35 represent the color values by which a normal observer recognizes a white object using the microscopy system according to the fifth embodiment, and the observation filter is shown in FIG. Configured such that the maximum spectral intensity of the light produced by the first light source is in each case about 10 times the magnitude of the maximum spectral intensity of the light produced by the second light source. And the maximum spectral intensity of the light produced by the first light source is about 6 times the magnitude of the maximum spectral intensity of the light produced by the third light source. Therefore, the non-fluorescent region of the object can be recognized as almost true color. In addition, the result of this configuration is that the fluorescent and non-fluorescent areas of the object are of approximately equal brightness.

The broken line shown in FIG. 13 represents the color of the non-fluorescent region of the object obtained by the change in the ratio between the intensity 52 of the second light (maximum thereof) and the intensity 53 of the third light (maximum thereof). In this way, a white point different from D50 can also be set advantageously.

Example of Sixth Embodiment FIG. 14 shows the wavelength-dependent transmittance 141 (solid line) of the observation filter 23 and the wavelength-dependent transmittance 142 (broken line) of the illumination filter 9 according to the sixth embodiment.

When λ1=350 nm to λ2=430 nm, the transmittance 141 of the observation filter 23 is about 0.005%. When λ3=435 nm to λ4=500 nm, the transmittance 141 of the observation filter 23 is about 2%. At approximately λ4 to λ5=560 nm, the transmittance 141 of the observation filter 23 is approximately 0.005%. At λ5 to λ6=750 nm, the transmittance 141 of the observation filter 23 is about 95%. Furthermore, W1=0.01%, W2=1% and W3=80% apply.

From 360 nm to approximately λ4, the transmittance 142 of the illumination filter 9 is about 95%. The transmittance 142 of the illumination filter 9 is approximately 0.005% at approximately λ4 to approximately λ5'=565 nm. From λ5′ to approximately λ6′=620 nm, the transmittance 142 of the illumination filter 9 is about 2%. The transmittance 142 of the illumination filter 9 is about 0.005% at about λ6′ to λ6.

The example illumination and observation filters for the sixth embodiment together have substantially the same effect as the example illumination and observation filters for the fifth embodiment. The observation filter 23 suppresses the first light reflected by the object 13, the first light having a high intensity substantially only at λ1 and λ2, and not substantially attenuated by the illumination filter 9 Through. Further, the second light reflected by the object 13 is restricted by the illumination filter and the observation filter to a wavelength substantially included only in λ3 to λ4. Further, the third light reflected by the object 13 is restricted by the illumination filter and the observation filter to a wavelength substantially included in only λ5′ to λ6′. Light in the wavelength range of approximately λ4 to λ5 is effectively suppressed by the illumination filter and the observation filter. Therefore, the non-fluorescent region of the object 13 can be observed as almost true color. Furthermore, the fluorescence emitted from the object 13 and having a high intensity substantially only at λ6′ to λ6 is transmitted by the observation filter 23, so that the fluorescence region of the object 13 can be observed with a reddish color value. ..

Example of Seventh Embodiment FIG. 15 shows illumination light spectra of the first, second and third light sources according to the seventh embodiment.

The first light source serves substantially only to excite the fluorescent dye fluorescein, ie to visualize the fluorescent region of the object. The second and third light sources serve substantially only to visualize the non-fluorescent areas of the object.

The first light source produces an illumination light spectrum 151 having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. Below about 430 nm and above about 500 nm, the intensity of the first light produced by the first light source is at most 1% of its maximum spectral intensity. The first light source is, for example, a light emitting diode.

The second light source produces an illumination light spectrum 152 having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. Below about 430 nm and above about 650 nm, the intensity of the second light produced by the second light source is at most 1% of its maximum spectral intensity. The second light source is, for example, a light emitting diode.

The third light source produces an illumination light spectrum 153 having a spectral emission maximum in the range of 610 nm to 640 nm and a full width at half maximum in the range of 10 nm to 20 nm. The third light source is, for example, a light emitting diode.

FIG. 16 shows the wavelength dependent transmittance 161 (solid line) of the observation filter 23 and the wavelength dependent transmittance 162 (broken line) of the illumination filter 9 according to the seventh embodiment.

When λ1=420 nm to λ2=480 nm, the transmittance 161 of the observation filter 23 is about 0.005%. When λ3=490 nm to λ4=500 nm, the transmittance 161 of the observation filter 23 is about 2%. At approximately λ4 to approximately λ5=540 nm, the transmittance 161 of the observation filter 23 is approximately 0.005%. At λ5 to λ6=700 nm, the transmittance 161 of the observation filter 23 is about 95%. At approximately λ6 to 750 nm, the transmittance 161 of the observation filter 23 is about 0.005%. Furthermore, W1=0.01%, W2=0.5% and W3=80% apply.

At 350 nm to approximately λ1, the transmittance 162 of the illumination filter 9 is about 0.005%. The transmittance 162 of the illumination filter 9 is approximately 95% at λ1 to approximately λ4. The transmittance 162 of the illumination filter 9 is approximately 0.005% at approximately λ4 to approximately λ5′=630 nm. The transmittance 162 of the illumination filter 9 is approximately 0.08% at approximately λ5′ to λ6′=690 nm. At approximately λ6′ to 750 nm, the transmittance 162 of the illumination filter 9 is approximately 0.005%.

Therefore, the observation filter 23 suppresses the first light reflected by the object 13, and the first light has a high intensity substantially only at λ1 and λ2. Further, the second light is suppressed by the illumination filter 9 when it exceeds λ4. The λ3 to λ4 part of the second light is reflected by the object 13 and transmitted by the observation filter 23, thus contributing to the visualization of the non-fluorescent region. Furthermore, the third light is suppressed by the illumination filter 9 below λ5'. The λ5′ to λ6′ portion of the third light is reflected by the object 13 and transmitted by the observation filter 23, thus contributing to the visualization of the non-fluorescent region. Furthermore, the fluorescence emitted from the object 13 and having a high intensity substantially only at λ5 to λ5′ is transmitted by the observation filter 23, so that the fluorescence region of the object 13 is observed with a yellowish color value. it can.

The first, second and third light intensities can be set individually (see FIG. 15), and by the specific selection of the transmittance 161 of the observation filter 23 and the transmittance 162 of the illumination filter 9. There are three main advantages. First, by appropriately setting the intensity of the light source, the fluorescent region and the non-fluorescent region can be recognized as having almost the same brightness. Furthermore, the light sources can now operate in their stable emission range, since the light produced by the second and third light sources is attenuated by the observation filter 23 and the illumination filter 9 to a sufficient extent. is there. Finally, as a result of being able to set the second and third light intensities separately, the color rendering of the non-fluorescent regions can be set such that the non-fluorescent regions are visible as nearly true color.

FIG. 17 shows a CIE 1931 xy standard chromaticity diagram including a spectrum locus S and a pure purple locus P. White circles represent the D50 white point where x=0.3457 and y=0.3585. The x-marks of approximately x=0.35 and y=0.33 represent the color values by which a normal observer perceives a white object using the microscopy system according to the seventh embodiment. Is configured as shown in FIG. 16, the maximum spectral intensity of the light generated by the first light source is the magnitude of the maximum spectral intensity of the light generated by the second light source in each case. Is about 3 times and the maximum spectral intensity of the light generated by the first light source is about 5 times the maximum spectral intensity of the light generated by the third light source. Therefore, the non-fluorescent region of the object can be recognized as almost true color. In addition, the result of this configuration is that the fluorescent and non-fluorescent areas of the object are of approximately equal brightness.

Third Aspect An example of a microscopy method according to the third aspect will be described with respect to FIGS. This method can be carried out by the microscopy system according to FIG. 1 and comprises the steps of generating illumination light, directing the generated illumination light at an object 13, and an observation beam path 22 for imaging the object 13 on an image plane 19. And generating a viewing filter 23 in the viewing beam path 22.

FIG. 18 illustrates one exemplary configuration of a microscopy system for performing a microscopy method for Protoporphyrin IX. The diagram 181 of FIG. 18 shows the wavelength-dependent absorption spectrum 184 of PpIX and the wavelength-dependent emission spectrum 185 of PpIX, which in each case is normalized to their respective maxima, see also the description relating to FIG. I want to. The diagram 182 of FIG. 18 shows the wavelength dependent transmittance 186 of the observation filter 23 for the same wavelength portion as the diagram 181. Diagram 183 shows the wavelength dependent intensity 187 of the illumination light directed at the object 13 for the same wavelength portion as diagram 181.

The first value W1 represents the average value of the transmittance 186 of the observation filter 23 over the first wavelength range WLB1. The second value W2 represents the average value of the transmittance 186 of the observation filter 23 over the second wavelength range WLB2. The third value W3 represents the average value of the transmittance 186 of the observation filter 23 over the third wavelength range WLB3. The first value W1 is less than the second value W2. The second value W2 is less than the third value W3.

The fourth value W4 represents the average value of the intensity 187 of the illumination light over the first wavelength range WLB1. The fifth value W5 represents the average value of the intensity 187 of the illumination light over the second wavelength range WLB2. The sixth value W6 represents the average value of the intensity 187 of the illumination light over the third wavelength range WLB3. The fourth value W4 is larger than the fifth value W5. The fifth value W5 is greater than the sixth value W6.

The first, second and third wavelength ranges do not overlap one another and are in each case 350 nm to 1000 nm. In the illustrated example, the first wavelength range WLB1 extends from about 370 nm to 430 nm, the second wavelength range WLB2 extends from about 470 nm to 580 nm, and the third wavelength range WLB3 extends from about 600 nm to 750 nm.

What is achieved with the values for the ratio of the first to sixth values specified for the third aspect is that the non-fluorescent region of the object is the light of the first wavelength range WLB1 and the second wavelength range WLB2. On the other hand, the fluorescence can be observed by the light in the third wavelength range WLB3. Furthermore, what is achieved on the basis of the stated ratio of the first to sixth values is that the non-fluorescent region and the fluorescent region can be recognized as almost equal brightness. What is further achieved is that non-fluorescent areas can be perceived as nearly true color.

FIG. 19 illustrates one exemplary configuration of a microscopy system for performing a microscopy method for fluorescein. Diagram 191 of FIG. 19 shows the wavelength-dependent absorption spectrum 194 of fluorescein and the wavelength-dependent emission spectrum 195 of fluorescein, in each case normalized to their respective maxima, see also the description relating to FIG. I want to. The diagram 192 of FIG. 19 shows the wavelength-dependent transmittance 196 of the observation filter 23 for the same wavelength part as the diagram 191. Diagram 193 shows the wavelength dependent intensity 197 of the illumination light directed at the object 13 for the same wavelength portion as diagram 191.

In the illustrated example, the first wavelength range WLB1 extends from about 440 nm to 500 nm, the second wavelength range WLB2 extends from about 640 nm to 750 nm, and the third wavelength range WLB3 extends from about 520 nm to 620 nm.

An example of an alternative microscopy method according to the third aspect is described with respect to FIGS. The method can be carried out by the microscopy system according to FIG. 1 and comprises the steps of generating illumination light, directing the generated illumination light at an object 13, and an observation beam path 22 for imaging the object 13 on an image plane 19. And generating a viewing filter 23 in the viewing beam path 22.

FIG. 20 shows a schematic view of a projected image of the object 13 on the image plane 19. The projected image of the object 13 includes a first position 201, a second position 202, and a third position 203. The light in the observation beam path 22 that produces a projected image in the image plane 19 has a color value in the first color value range 211 at the first position 201. The light in the observation beam path 22 that produces the projection image in the image plane 19 has a color value in the second color value range 212 at the second position 202. The light in the observation beam path 22 that produces the projected image in the image plane 19 is at a third position 203 outside the first color value range 211 and outside the second color value range 212. Has a value.

FIG. 22 shows a CIE 1931 standard chromaticity diagram for explaining the first color value range 211, the second color value range 212, and the third color value range 213. The first color value range 211 comprises the red value, ie the color value of the intense fluorescence of the fluorescent dye PpIX. The second color value range 212 includes blue values, that is, color values that can efficiently excite the fluorescent dye PpIX. The third color value range 213 includes the remaining color values of the visible color values that are not included in the first and second color value ranges. The arrow 214 indicates the minimum color difference between the first color value range 211 and the second color value range 212.

According to an alternative microscopy method, the illumination light is generated such that the ratio between the first characteristic value and the second characteristic value has a value within a predetermined, limiting numerical range. The first characteristic value represents the value of a characteristic variable relating to the light intensity (or brightness, luminous flux, etc.) of the observation beam path 22 at the first position 201. The second characteristic value represents the same characteristic variable for light in the observation beam path 22 at the second location 202.

For example, the first feature value represents an average value of the light intensity of the observation beam path 22 at the first position 201, and the second feature value is the light intensity of the observation beam path 22 at the second position 202. Represents the average value of. When the illumination light is generated such that the ratio between the first feature value and the second feature value is in the numerical range of 20/1 to 1/20 or in the numerical range narrower than that, the first position 201, That is, the fluorescent region of the object 13 and the second position 202, that is, the non-fluorescent region of the object 13 can be recognized as having substantially the same brightness.

An example of a microscopy method is described below with respect to FIG. This example relates to the method of controlling the microscopy system of FIG. 1, where the brightness of the fluorescent and non-fluorescent areas of the object 13 are matched.

In step S1, the image of the object 13 in the image plane 19 is recorded by the color image detector 20. The image is an image of a projected image of the object 13 generated in the image plane 19 through the observation beam path 22.

21 shows a schematic diagram of the image 205 recorded in step S1, which is an image of a projected image of the object 13 on the image plane 19 shown in FIG. The image 205 consists of multiple image points 206.

In step S2 following step S1, the first image point 207 is identified from the image point 206. The first image point 207 corresponds to the first position 201. Image 205 has color values that are within first color value range 211 at first image point 207. The first image point 207 is specified, for example, by comparing the color value at the image point 206 of the image 205 with a predetermined first color value range 211.

In step S3 following step S1, the second image point 208 is identified from the image point 206. The second image point 208 corresponds to the second position 202. Image 205 has color values that are within second color value range 212 at second image point 208. The second image point 208 is identified, for example, by comparing the color value of the image point 206 of the image 205 with a predetermined second color value range 212.

Following step S2, in step S4, a first feature value is identified based on the intensity of the image 205 at the first image point 207. For example, the average value or the maximum value of the intensity at the first image point 207 is specified.

Following step S3, in step S5, a second feature value is identified based on the intensity of the image 205 at the second image point 208. For example, the average value or the maximum value of the intensity at the second image point 208 is specified.

In step S6, which follows steps S4 and S5, the illumination light is observed beam path 22 in the image plane 19 depending on the first and second characteristic values or at the first and second positions 201, 202. Is set according to the intensity of the light or the intensity at the first and second image points 207 and 208. That is, the wavelength-dependent intensity distribution of the illumination light is changed. In this case, the illumination light is generated so that the above-mentioned ratio of the first and second characteristic values has a value within a predetermined numerical range.

The method can be performed iteratively, which is indicated by the arrow from step S6 to step S1 in FIG.

DESCRIPTION OF SYMBOLS 1 Microscopic inspection system 3 Illumination device 5 Light source 7 Illumination optical unit 9 Illumination filter 10, 22 Observation beam path 11 Object area 12 Controller 13 Object 14 Microscopic inspection optical unit 15 Objective lens 16, 17 Lens 18 Object plane 19 Image plane 20 Optical detection Instrument 21 Signal 23 Observation filter 31, 32, 51, 151-153 Illumination light spectrum 41, 61, 81, 101, 102, 121, 141, 142, 161, 162, 186, 196 Wavelength dependent transmittance 52 Second light Intensity 53 third intensity of light 181-183, 191-193 formula 184, 194 wavelength-dependent absorption spectrum 185, 195 wavelength-dependent emission spectrum 187, 197 wavelength-dependent intensity 201 first position 202 second position 203 third Position 205 image 206 image point 207 first image point 208 second image point 211 first color value range 212 second color value range 213 third color value range 214 arrow

Claims (39)

A microscopy system (1) for simultaneously observing fluorescent and non-fluorescent regions of an object (13),
A microscopy optics unit (14) configured to image the object plane (18) onto the image plane (19) through the observation beam path (22);
An observation filter (23) positionable in the observation beam path (22),
The transmittance (41, 61, 81, 101, 121, 141, 161) of λ1 to λ2 of the observation filter (23) is less than W1, larger than W2 at λ3 to λ4, and larger than W3 at λ5 to λ6. big,
λ1, λ2, λ3, λ4, λ5 and λ6 are wavelengths to which 350 nm<λ1<λ2≦λ3<λ4≦λ5<λ6<750 nm are applied,
W1, W2, W3 are observation filters (23), which are values for which 0<W1<W2<W3<1 applies,
a first light source (5) configured to generate a first light having a wavelength of λ1 to λ2 and direct it toward the object plane (18);
a second light source (5) configured to generate a second light having a wavelength of λ3 to λ4 and direct it to the object plane (18);
A controller (12) configured to individually control the first light source and/or the second light source so that the intensity of the first light and the intensity of the second light are The ratio of is variable, the microscopy system (1) including a controller (12). 350 nm<λ1<400 nm and/or 410 nm<λ2<440 nm, and/or 440 nm<λ3<460 nm and/or 480 nm<λ4<620 nm, and/or 530 nm<λ5<620 nm and/or 650 nm<λ6<750 nm, and/or Or W1=0.1% or W1=0.01%, and/or W2=1%, and/or W3=90%
The microscopy system according to claim 1, wherein The first light source (5) is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 400 nm to 420 nm and a full width at half maximum in the range of 10 nm to 20 nm, and/or the second light source. The microscopy system according to claim 1 or 2, wherein (5) is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. 480 nm<λ4<520 nm, and/or the transmittance (41) of λ3 to λ4 of the observation filter (23) is greater than 10% and less than 60%, and/or the observation filter (23). The transmittance (41) of λ2+Δ to λ3-Δ and λ4+Δ to λ5-Δ is less than 0.1 times W1, 3 nm≦Δ≦10 nm is applicable, and/or the first transmittance above λ3. The intensity (31) of the light is at most 1% of its maximum spectral intensity, and/or the intensity (32) of the second light below λ2 and above λ5 is the maximum of its maximum spectral intensity. The microscope inspection system according to any one of claims 1 to 3, wherein the microscope inspection system is 1%. further comprising a third light source (5) configured to generate a third light having a wavelength of λ7 to λ8 and direct it to said object plane (18), where λ7, λ8 are wavelengths , And [lambda]2<[lambda]7<[lambda]8<[lambda]5. 4. The microscopy system according to claim 1, wherein: The microscope according to claim 5, wherein the third light source (5) is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. Inspection system. The transmittance (61) of λ3 to λ6 of the observation filter (23) is greater than W3 and/or the intensity (51) of the first light above λ3 is at its maximum spectral intensity maximum. 1% and/or the intensity of the second light (52) below λ2 and above λ5 is at most 1% of its maximum spectral intensity and/or below λ2 and above λ5. The microscopy system according to claim 5 or 6, wherein the third light intensity (53) is at most 1% of its maximum spectral intensity. The transmittance (81, 101) of λ3 to λ4 of the observation filter (23) is less than 5%, 600 nm<λ4<λ5<620 nm is applied, and/or the first light above λ3. The intensity (51) is at most 1% of its maximum spectral intensity, and/or the intensity (52) of the second light below λ2 and above λ5 is at most 1 of its maximum spectral intensity. %, and/or the intensity (53) of the third light below λ2 and above λ5 is at most 1% of its maximum spectral intensity. Further comprising an illumination filter (9) positionable between the light source (5) and the object plane (18),
The transmittances (102) of λ1 to λ3′ and λ4′ to λ4 of the illumination filter (9) are greater than W3, and less than W1 at λ3′+Δ to λ4′−Δ and λ4+Δ to λ6, and 3 nm≦. Δ≦10 nm applies,
λ3′, λ4′ are wavelengths, and λ3<λ3′<λ4′<λ4, and 480 nm<λ3′<520 nm, and 520 nm<λ4′<550 nm, and λ4′−λ3′>20 nm, and / Or the intensity (51) of the first light above λ3 is at most 1% of its maximum spectral intensity and/or the intensity of the second light below λ2 and above λ4'. (52) is at most 1% of its maximum spectral intensity, and/or said intensity (53) of said third light at λ4′ to λ4 is large and below λ3′ and above λ5. The microscopy system according to claim 8, wherein the maximum spectral intensity is at most 1%. The transmittance (121) of λ3 to λ4 of the observation filter (23) is less than 5%,
The transmittance (121) of λ7 to λ8 of the observation filter (23) is greater than W2 and less than 5%,
The transmittance (121) of λ4+Δ to λ7−Δ of the observation filter (23) is less than W1, and 3 nm≦Δ≦10 nm applies,
λ7, λ8 are wavelengths, λ4<λ7<λ8<λ5, and 480 nm<λ4<520 nm, and 520 nm<λ7<550 nm, and λ7−λ4>20 nm, and λ5-λ8<30 nm, and/or The intensity (51) of the first light above λ3 is at most 1% of its maximum spectral intensity and/or the intensity (52) of the second light below λ2 and above λ7. Is at most 1% of its maximum spectral intensity, and/or the intensity of said third light (53) below λ4 and above λ5 is at most 1% of its maximum spectral intensity. The microscope inspection system according to 5 or 6. The transmittance (141) of λ3 to λ4 of the observation filter (23) is less than 5%,
The transmittance (141) of λ4+Δ to λ5-Δ of the observation filter (23) is less than W1.
480 nm<λ4<520 nm, and 530 nm<λ5<560 nm, and λ5-λ4>20 nm, and/or the microscopy system (1) is between the light source (5) and the object plane (18). Further comprising a positionable lighting filter (9),
The transmittance (142) of λ1 to λ4 of the illumination filter (9) is larger than W3,
The transmittance (142) of λ4+Δ to λ5′-Δ of the illumination filter (9) is less than W1;
The transmittance (142) of λ5′ to λ6′ of the illumination filter (9) is greater than W2 and less than 5%,
The transmittance (142) of λ6′ to λ6 of the illumination filter (9) is less than W1, and 3 nm≦Δ≦10 nm applies,
λ5′, λ6′ are wavelengths, and λ4<λ5′<λ6′<λ6 holds,
530 nm<λ5′<570 nm and 600 nm<λ6′<620 nm apply, and/or the intensity (51) of the first light above λ3 is at most 1% of its maximum spectral intensity, and/or Or the intensity (52) of the second light below λ2 and above λ5 is at most 1% of its maximum spectral intensity, and/or the intensity of the third light below λ4 and above λ5. The microscope inspection system according to claim 5 or 6, wherein (53) has a maximum spectral intensity of 1%. The microscopy system according to any one of claims 1 to 8 or 10, wherein there is no illumination filter between the object plane (18) and the light source (5). a third light source (5) configured to generate a third light having a wavelength of λ5′ to λ6′ and direct it toward the object plane (18),
Illumination filter (9) which can be arranged between the light source (5) and the object plane (18)
Further including,
The transmittance (162) of λ1 to λ4 of the illumination filter (9) is larger than W3,
The transmittance (162) of λ5′ to λ6′ of the illumination filter (9) is greater than W2 and less than 5%,
λ5′, λ5′ are wavelengths, and λ5<λ5′<λ6′ holds,
The microscopy system according to claim 1, wherein the transmittance (161) of λ3 to λ4 of the observation filter (23) is less than 5%. The transmittance (162) of λ4+Δ to λ5′+Δ of the illumination filter (9) is less than W1, 3 nm≦Δ≦10 nm applies, and/or the λ6′ to 750 nm of the illumination filter (9). The transmittance (162) is less than W1, and/or the transmittance (161) of λ4+Δ to λ5-Δ of the observation filter (23) is less than W1, and 3 nm≦Δ≦10 nm is applied. Item 13. The microscope inspection system according to Item 13. 350 nm<λ1<460 nm and/or 460 nm<λ2<480 nm, and/or 470 nm<λ3<490 nm and/or 490 nm<λ4<510 nm, and/or 520 nm<λ5<550 nm and/or 680 nm<λ6<750 nm, and/or Or 620 nm<λ5′<650 nm and/or 680 nm<λ6′<750 nm, and/or W1=0.1% or W1=0.01%, and/or W2=0.5%, and/or W3=90 %, and/or the intensity (151) of the first light above λ5 is at most 1% of its maximum spectral intensity, and/or the second light above λ3 and above λ5′. Said intensity (152) of at most 1% of its maximum spectral intensity and/or said third light intensity (153) below λ4 is at most 1% of its maximum spectral intensity. The microscope inspection system according to claim 13 or 14. The first light source (5) is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 440 nm to 460 nm and a full width at half maximum in the range of 10 nm to 20 nm, and/or the second light source. (5) is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 500 nm to 550 nm and a full width at half maximum in the range of 40 nm to 110 nm, and/or the third light source (5) is 16. The microscopy system according to any one of claims 13 to 15, which is configured to generate a spectral intensity distribution having a spectral emission maximum in the range of 600 nm to 640 nm and a full width at half maximum in the range of 10 nm to 20 nm. The controller (12) individually sets at least one operating current and/or operating voltage of the light source (5), whereby the intensity (31, 31, of the light generated by the light source (5) is set. 32, 51, 52, 53, 151, 152, 153) is configured individually to provide the microscopy system according to any one of claims 1 to 16. A method of operating a microscopy system, in particular a microscopy system (1) according to any one of claims 1 to 17,
Generating illumination light and directing the generated illumination light at an object (13),
Generating an observation beam path (22) for imaging said object (13) on an image plane (19), an observation filter (23) being arranged in said observation beam path (22),
The illumination light is

Is generated so that

Represents the color position of the white point in the CIE standard chromaticity diagram of the CIE 1931 standard color system,

Represents a color position having coordinates x _f , y _f in the CIE standard chromaticity diagram of the CIE 1931 standard color system, and the coordinates x _f , y _f are

as well as

Where X, Y and Z are

Represents the tristimulus values of the CIE 1931 standard color system defined by
I(λ) represents the intensity of the illumination light,
T(λ) represents the transmittance (41, 61, 81, 101, 121, 141, 161) of the observation filter (23),

Represents the spectral value function of the CIE 1931 standard color system, and k is a constant method.

,Especially

19. The method of claim 18, wherein A microscopy method,
Generating illumination light and directing the generated illumination light at an object (13),
Generating an observation beam path (22) for imaging said object (13) on an image plane (19), an observation filter (23) being arranged in said observation beam path (22),
The first value (W1), which represents the average value of the transmittance (186, 196) of the observation filter (23) over the first wavelength range (WLB1), is the first value (W1) over the second wavelength range (WLB2). 23) less than a second value (W2) representing the average value of the transmittances (186, 196),
The second value (W2) is less than a third value (W3) representing an average value of the transmittances (186, 196) of the observation filter (23) over a third wavelength range (WLB3),
A fourth value (W4) representing an average value of the intensities (187, 197) of the illumination light over the first wavelength range (WLB1) is the intensity of the illumination light over the second wavelength range (WLB2). Greater than the fifth value (W5), which represents the average value of (187, 197),
The fifth value (W5) is larger than a sixth value (W6) representing an average value of the intensities (187, 197) of the illumination light over the third wavelength range (WLB3),
The microscopy method, wherein the first, second and third wavelength ranges do not overlap each other and in each case are 350 nm to 1000 nm. The first wavelength range (WLB1) is at least 5%, in particular at least 10%, the absorptance (184, 194) of the fluorescent dye present in the object (13)-normalized to its maximum-, More particularly including wavelengths that are at least 50%,
Said third wavelength range (WLB3) comprises wavelengths in which the emission rate (185,195) of the fluorescent dye present in said object (13) is at least 5%, in particular at least 10%, more particularly at least 50%. The microscopic inspection method according to claim 20. In the second wavelength range (WLB2), the absorptance (184, 194) of fluorescent dyes present in the object (13)-normalized to its maximum value-is at most 20%, especially at most 10 %, more particularly up to 5%, and the luminescence rate (185, 195) of the fluorescent dye present in the body (13)-normalized to its maximum value-up to 20%, especially up to 22. The microscopy method according to claim 20 or 21, comprising only wavelengths which are 10%, more particularly at most 5%. The first wavelength range (WLB1) includes a wavelength of 405 nm, and particularly includes a wavelength of 390 nm to 420 nm,
The second wavelength range (WLB2) includes a wavelength of 450 nm to 600 nm,
The microscopy method according to any one of claims 20 to 22, wherein the third wavelength range (WLB3) includes a wavelength of 620 nm to 720 nm. The first wavelength range (WLB1) includes a wavelength of 480 nm to 500 nm,
The second wavelength range (WLB2) includes wavelengths of 620 nm to 750 nm,
The microscope inspection method according to any one of claims 20 to 22, wherein the third wavelength range (WLB3) includes a wavelength of 550 nm to 600 nm. The ratio between the first value (W1) and the second value (W2) is at most 1/100, in particular at most 1/1000, and/or the second value (W2) and the The ratio with the third value (W3) is at most 0.9, especially at most 0.8, more especially at most 0.5, and/or the fourth value (W4) and the fifth value. To a value (W5) of at least 2, in particular at least 5, and/or the ratio of said fourth value (W4) to said sixth value (W6) is at least 1000, in particular at least 10000. The microscope inspection method according to any one of claims 20 to 24. The illumination light is generated using a plurality of narrow band light sources (5), at least one of the light sources (5) generating light having a wavelength within the first wavelength range (WLB1), 26. The microscopy method according to any one of claims 20 to 25, wherein at least one of the light sources (5) produces light having a wavelength within the second wavelength range (WLB2). 27. The microscopy method according to claim 26, wherein the light source (5) substantially does not generate light having a wavelength within the third wavelength range (WLB3). A microscopy method,
Generating illumination light and directing the generated illumination light at an object (13),
Creating an observation beam path (22) that images the object (13) onto an image plane (19),
The illumination light is generated such that the ratio between the first characteristic value and the second characteristic value has a value in the range of 20/1 to 1/20,
The first characteristic value is the value of a characteristic variable relating to the light intensity of the observation beam path (22) at the first position (201) in the image plane (19), and at the first position , The color value of the light of the observation beam path (22) is within a first color value range (211) of the color space,
The second characteristic value is the value of a characteristic variable relating to the light intensity of the observation beam path (22) at a second position (202) in the image plane (19), and the second position Then, the microscopy method, wherein the color value of the light of the observation beam path (22) is within a second color value range (212) of the color space. The first characteristic value represents a maximum or average value of the intensities of the light in the observation beam path (22) at the first position (201),
29. The microscopy method according to claim 28, wherein the second characteristic value represents a maximum or average value of the intensity of the light of the observation beam path (22) at the second position (202). Identifying the first position (201) in the image plane (19), wherein the color value of the light of the observation beam path (22) is the color space. Within the first color value range (211) of
Identifying the second position (202) in the image plane (19), wherein the color value of the light in the observation beam path (22) is the color space. Within the second color value range (212) of
The illumination light is dependent on the intensity of the light in the observation beam path (22) at the identified first position (201) and at the identified second position (202). 30. The microscopy method according to claim 28 or 29, generated according to the intensity of the light of the path (22). Recording an image (205) of the object (13) in the image plane (19), the image (205) comprising a plurality of image points (206);
Comparing the color value at the image point (206) of the image (205) with the first color value range (211) and/or the second color value range (212);
Identifying a first image point (207) from the plurality of image points (206) based on the result of the comparison, wherein the first image point (207) is within the image plane (19). Corresponding to the first position (201) of the color value at the first image point (207) is within the first color value range (211) of the color space;
Identifying a second image point (208) from the plurality of image points (206) based on the result of the comparison, the second image point (208) being within the image plane (19). Corresponding to the second position (202) of the color value at the second image point (208) is within the second color value range (212) of the color space. The microscopy method according to claim 30. Identifying the first characteristic value based on the intensity of the light of the observation beam path (22) at the identified first position (201);
Identifying the second characteristic value based on the intensity of the light of the observation beam path (22) at the identified second position (202).
The microscopy method according to claim 30 or 31, wherein the illumination light is generated according to the specified first characteristic value and according to the specified second characteristic value. The illumination light is generated by a plurality of light sources (5) that generate light in different wavelength ranges,
The step of generating the illuminating light sets the energy supplied to at least one of the plurality of light sources (5) for generating the illuminating light according to the first and second characteristic values, in particular 33. The microscopy method according to any one of claims 28 to 32, including a step of changing. The first color value range (211) and the second color value range (212) do not overlap and/or the first color value range (211) in the CIE 1976 u'v' chromaticity diagram. ) And the second color value range (212) has a minimum color difference (214) of at least 0.01. 34. A microscopy method according to any one of claims 28 to 33. The color value range (211) is limited to color values having a maximum color difference of 0.1 with respect to a reference color value in the CIE 1976 u'v' chromaticity diagram,
The reference color value is at least 5%, in particular at least 10%, more in particular at least 50% of the emission rate (185,195) of the fluorescent dye present in the object (13)-normalized to its maximum value. 35. The microscopy method according to any one of claims 28 to 34, which corresponds to light within a reference wavelength range including only a wavelength of. The first color value range (211) includes color values corresponding to light having a wavelength of 635 nm, and/or the first color value range (211) is a CIE 1976 u'v' chromaticity diagram. 36. The microscopy method according to any one of claims 28 to 35, wherein the microscopy is limited to color values having a maximum color difference of 0.1 with respect to a reference color value corresponding to light having a wavelength of 630 nm. The first color value range (211) includes color values corresponding to light having a wavelength of 530 nm, and/or the first color value range (211) is a CIE 1976 u'v' chromaticity diagram. 36. The microscopy method according to any one of claims 28 to 35, wherein the microscopy is limited to color values having a maximum color difference of 0.1 with respect to a reference color value corresponding to light having a wavelength of 530 nm. The first color value range (211) in the CIE 1976 u'v' chromaticity diagram has the coordinates u'=0.113 and v'=0.575 or the coordinates u'=0.570 and v. 38. Microscopic examination according to any one of claims 28 to 37, comprising a color value having'=0.514 and/or the second color value range (212) comprising a white point D50 or D65. Method. The illumination light is generated regardless of the light intensity of the observation beam path (22) at the third position (203) in the image plane (19), and the observation beam at the third position (203) is generated. 39. A microscopy method according to any one of claims 28 to 38, wherein the color value of the light of the path (22) is outside the first and second color value range.

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